EVALUATION OF TECHNIQUES FOR THE CRYOPRESERVATION OF WASHED SPERMATOZOA - COMPARISONS BETWEEN HAMS F-10 AND TEST-YOLK MEDIA

The objective of this study was to develop new techniques for the cryo preservation of washed spermatozoa. Two media (Ham's F-10 and nontherm oprecipitated TEST-yolk buffer [NT-TYB]) containing 7% (v/v) glycerol were compared to semen cryopreservation by adding glycerol directly to the semen. Twenty four men collected a semen specimen each after 4 da ys of sexual abstinence via the use of a semen collection device at in tercourse. Specimens were assessed for volume (ml), count (x10(6)), pe rcentage and grade of motility, morphology (% normal) and acrosomal st atus (% intact acrosomes). Each ejaculate was split into 3 aliquots (A liquots 1 to 3) and processed for freezing. Aliquot 1 was prepared for cryo preservation by adding glycerol (7% [v/v] final concentration) d irectly via a dropwise mode. Aliquot 2 and 3 were diluted 1:1 (v/v) wi th Ham's F-10 and NT-TYB, respectively. Aliquots 2 and 3 were then cen trifuged (400 x g for 10 minutes) and resuspended into the correspondi ng media containing 7% (v/v) glycerol to complete the sperm wash proce dure. All aliquots were frozen in 0.5 mi french straws. Sperm specimen s were frozen in liquid nitrogen (LN2) vapor from +23 degrees C to -68 degrees C at a slow rate (2.3 degrees C/minute), after which the spec imens mere plunged directly into LN2 and stored for 30 days. The quali ty of the spermatozoa were monitored throughout each step of the overa ll procedure by measuring the motility characteristics of the spermato zoa. Straws corresponding to each aliquot were thawed in a mater bath at 37 degrees C for 2 minutes, followed by assessment of sperm motilit y and acrosomal status. The percentage of motility after thawing was 3 1.6+/-5.6%, 32.8+/-1.8% and 37.3+/-1.9% in Aliquots 1 to 3, respective ly. Similarly, the grade of motility was 2.4+/-0.2, 2.6+/-0.1 and 3.0/-0.1 in Aliquots 1 to 3, respectively. The acrosomal status (% intact acrosomes) in Aliquots 1 to 3 was 41.2+/-2.6, 43.1+/-3.6 and 51.6+/-4 .5, respectively. The results suggest that the characteristics of sper matozoa washed and frozen in NT-TYB (Aliquot 3) were improved over tho se spermatozoa prepared via direct addition of glycerol to the semen ( Aliquot 1) or by using Ham's F-10 (Aliquot 2). The most significant re duction noted during freezing was in the loss of acrosomal integrity. The results obtained in this study point out that washed spermatozoa c an be cryopreserved with some success and that the recovered spermatoz oa could be used for intrauterine insemination in an artificial insemi nation program using husband's or donor sperm, or for the various assi sted reproductive technology procedures. It is the opinion of the auth ors that the information generated in this study is of importance for those scientists and clinicians involved in the handling and manipulat ion of cryopreserved spermatozoa. (C) 1998 Tohoku University Medical P ress.